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Research article
S yn thesis and A n tic a n c e r A c tiv ity of N e w Thiopyrano[2,3-£/]thiazoles Based on C innam ic Acid Am ides Andrii Lozynskyi, Borys Z imenkovsky, Roman Lesyk * Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010, Lviv, Ukraine. * Corresponding author. E-mail: [email protected] (R. Lesyk) Sei Pharm. 2014; 82: 723-733
doi:10.3797/scipharm.1408-05
Published: Accepted:
Received:
September 15ih 2014 September 15th 2014
August 5th 2014
This article is available from: http://dx.doi.org/10.3797/scipharm.1408-05 © Lozynskyi et air, licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.Org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract________________________________________________ Novel /'e/-(5R,6S,7S)-2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2/-/-thiopyrano [2,3-d]thiazole-6-carboxylic acid amides were synthesized in a hetero-D\e\sAlder reaction with a series of cinnamic acid amides. The synthesized compounds were tested for their anticancer activity in vitro in the standard National Cancer Institute 60 cancer cell line assay. Promising compounds 3e, 3g, and 3h with moderate antitumor activity were identified among the synthesized series.
Keywords hefero-Diels-Alder reaction • Cynnamic acid amides Thiopyrano[2,3-cf|[1,3]thiazoles • Anticancer activity
• 5-Ylideneisorhodanines
•
Introduction Investigations of thiopyrano[2,3-c/]thiazole derivatives, the isosteric mimics of biologically active 5-ylidene-4-thiazolidinones, led to the synthesis of compounds with anticancer, antitrypanosomal, and antimycobacterial properties which can provide an opportunity to further study and explore the pharmacological activity of these heterocyclic systems in the future [1-13]. We decided to combine in a single heterocyclic system the thiazolidinone moiety and a fragment of cinnamic acid (Sch. 1). Cinnamic acid and its derivatives exhibit antitumor, antimicrobial, antifungal action and act as histamine H3-receptor antagonists
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[14-16]. Consequently, we have synthesized thiopyrano[2,3-cf]thiazoles using cinnamic acid amides as the dienophile in the reaction of hefero-Diels-Alder. In addition, heterodiene synthesis allows the fixing of the biologically important 4-thiazolidinone fragment in a rigid fused system, preserving its biological activity. Moreover, the combination of thiazole and thiopyran in a fused heterosystem is a precondition for creating ligand-target binding and enhances the potential selectivity to biotargets. This approach suggests the critical impact of the substituent on the biological activity with particular selectivity to various cancer cell lines. Cinnamic acid amides moiety Structure of target compounds
5-Arylidene-4-thiazolidinone moiety
H
Zelisko et al., 2012 [11] Sch. 1 .
0
Anticancer and antimycobacterial activities
Anticancer activity
Atamanyuk et al., 2013 [10]
Lesyk et al., 2006 [4]
Background for the synthesis of target compounds
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Results and Discussion Chemistry The starting 5-aryl(hetaryl)idene-4-thioxo-2-thiazolidinones 1a-d were obtained by the treatm ent of 4-thioxo-2-thiazolidinone with the appropriate aldehydes in glacial acetic acid with a catalytic am ount o f fused sodium acetate [4, 12]. The cinnam ic acid amides were synthesized by the interaction o f the corresponding cinnam ic acid chloride with 4-substituted anilines, morpholine, and 2-am inopyridine in anhydrous dioxane. The heteroDiels-Alder reaction of 2 a -f with 5-aryl(hetaryl)idene-4-thioxo-2-thiazolidinones 1a-d yielded a series of novel re/-(5R,6S,7S)-2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2/-/thiopyrano[2,3-cf]thiazole-6-carboxylic acid amides (Sch. 2).
1a R = 4-OH-C6H4 3a R
1b R = 4-CI-C6H4 1c R = 4-Me-C6H4 1d thiophen-2-yl
4-OH-C6H4, R1= Cl
3b R
4-CI-C6H4, R1 = OH
3c R
4-Me-C6H4, R1 = S 0 2NH2
3d R
4-CI-C6H4, R1 = S 0 2NH2
3e R
4-CI-C6H4, R1 = Cl
3f R
4-Me-C6H4, R1 = Cl
3g R
4-CI-C6H4, R1 = Me
3h R
4-Me-C6H4, R1 = Me
3i R
thiophen-2-yl, R1 = Cl
3j R = 4-CI-C6H4 3I
R = 4-CI-C6H4
3k R = thiophen-2-yl
3m R = 4-Me-C6H4
Sch. 2.
Synthesis of 2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2/-/-thiopyrano[2,3-d]thiazole-6-carboxylic acid amides
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The structure of the synthesized compounds was confirmed by 1H- and 13C NMR. We found the features of the stereochemistry of the above Ziefero-Diels-Alder reaction. Particularly, we have observed that cinnamic acid amides in the [4+2]-cyclocondensation of 5-arylideneisorhodanines form a pair of re/-(5R,6S,7S)-diastereomers. This claim is based on the coupling constant values within 10.4-11.5 Hz and the spectral signals of the thiopyran fragment (triplet and two doublets at 3.40-4.87 ppm), which prove an axial-axial interaction of 5-H, 6-H and 6-H, 7-H proton pairs. Importantly, a similar pattern was observed earlier for cinnamic acids as the dienophile in the reactions of befero-Diels-Alder [
11, 12].
Tab. 1.
Cytotoxic activity of the tested compounds in the concentration 10 60 cancer cell lines
Test Average cpds. growth, % 3b
82.76
3c
101.41
3e
57.09
3g
57.89
3h
77.68
3i
80.64
3j 3k
88.76
3I
100.81
3m
96.07
95.84
Range of growth, %
M against
Most sensitive cell line growth, % (cancer //ne/type) 53.05 (RPMI-8226 / leukemia) 53.05-106.26 61.05 (SF-295 / CNS cancer) 88.61-117.37 88.61 (RXF 393 / renal cancer) 27.11 (MOLT-4 / leukemia) 26.38 (HCT-116 / colon cancer) 32.89 (SF-295 / CNS cancer) 26.38-94.10 35.53 (PC-3 / prostate cancer) 33.44 (MCF7 / breast cancer) 33.81 (T-47D1 breast cancer) 26.51 (MOLT-41 leukemia); 37.02 (RPMI-82261 leukemia) 39.39 (A549/ATCC1 non-small cell lung cancer) 32.09 (HCT-1161 colon cancer) 26.51-91.71 33.18 (SF-2951 CNS cancer) 33.99 (PC-31 prostate cancer) 31.77 (M C F71 breast cancer) 39.88 (T-47D / breast camcer) 30.79 (HOP-921 non-small cell lung cancer) -42.92 (NCI-H5221 non-small cell lung cancer) -42.92-114.10 35.63 (SK-MEL-51 melanoma) -21.73 (CAKI-11 renal cancer) 37.39 (UO-311 renal cancer) 57.75 (SF-295/ CNS cancer) 51.43-119.84 51.43 (PC-3 / prostate cancer) 59.03 (MCF7 / breast cancer) 61.65-112.27 61.65 (SNB-75 / CNS cancer) 72.63-120.48 72.63 (T-47D / breast cancer) 77.27 (SNB-75 1 CNS cancer) 73.58-120.80 78.29 (UO-31 1 renal cancer) 73.58 (T-47D 1 breast cancer) 73.69-110.93 73.69 (SR 1 leukemia);
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Biological Activity The synthesized re/-(5R,6S,7S)-2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2H-thiopyrano[2,3-d]thiazole-6-carboxylic acid amides were selected by the National Cancer Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov) for the in vitro cell line screening to investigate their anticancer activity. Anticancer assays were performed according to the NCI protocol, which is described elsewhere [5-7, 17]. The compounds were evaluated for antitumor activity against 60 cancer lines at a 10 pM concentration. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. The screening results are shown in Table 1. The tested compounds showed different levels of activity on various cancer cell lines. The most active compounds were 3e, 3g, 3h, being highly potent in certain lines of cancer, but they had almost no activity in others. Compounds 3e, 3g have a selective effect on the growth of MOLT-4 (leukemia), HCT-116 (colon cancer), SF-295 (CNS cancer), PC-3 (prostate cancer), MCF7, and T-47D (breast cancer) cancer cell lines in comparison with others.
Sch. 3.
SAR of anticancer potency of the synthesized thiopyrano[2,3-d]thiazole-6carboxylic acids amides
The empirical SAR study (Sch. 3) revealed that: (1) the anticancer activity of the synthesized compounds is sensitive to the nature of the amide fragment in position 6 and substitution in position 7 of the thiopyrano[2,3-d]thiazole moiety;
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(2) introduction of p-Me- or p-CI-C6H4 groups in the amide fragment enhances the potency; (3) the loss of anticancer activity is caused by the introduction of morpholin and pyridine fragments in position 6 or substitution of the arylamide moiety by OH or sulfanilamido groups; (4) synthesized thiopyrano[2,3-c/]thiazole-6-carboxylic acid amides with p-Me- and P-CI-C6H4 groups in postion 7 have the most preferable level of activity compared to other derivatives.
Experimental Chemistry All materials were purchased from Merck, Sigma-Aldrich, or Lancaster and were used without purification. 5-Aryl(hetaryl)idene-4-thioxo-2-thiazolidinones 1a-d were employed as starting materials and prepared according to the method described previously [4, 12]. Melting points were determined in open capillary tubes and were uncorrected. The elemental analyses (C, H, N) were performed using the Perkin-Elmer 2400 CHN analyzer and were within 0.4% of the theoretical values. The 1H- and 13C NMR spectra were recorded on the Varian Gemini 400 MHz or Bruker 125 MHz for frequencies of 100 MHz in DMSO-de using tetramethylsilane as an internal standard. Chemical shifts are reported in ppm units with the use of a 5 scale. The purity of all obtained compounds was checked by 1H-NMR and TLC. General Procedure of the Hetero-Diels-Alder Reaction Affording 3a-m A mixture of appropriate 5-aryl(hetaryl)idene-4-thioxo-2-thiazolidinone (5 mmol) and cinnamic acid amide (5.5 mmol) was refluxed for 4-7 h with a catalytic amount of hydroquinone (2-3 mg) in 15 ml of glacial acetic acid and left overnight at room temperature. The obtained solid products were collected by filtration, washed with water, methanol (5-10 ml), diethyl ether, and recrystallized from the appropriate solvent. rel-(5R, 6S, 7S)-N-(4-Chlorophenyl)-7-(4-hydroxyphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3a) Yield: 59%, mp 234-236°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.47 (t, 1H, J = 10.4 Hz, 6-H), 4.21 (d, 1H, J = 10.4 Hz, 7-H), 4.83 (d, 1H, 10.4 Hz, 5-H), 6.70 (d, 2H, J = 8.8 Hz, arom.), 7.00 (d, 2H, J = 8.8 Hz, arom.), 7.12 (d, 2H, J = 8.8 Hz, arom.), 7.20 (t, 1H, J = 7.2 Hz, arom.), 7.28 (t, 2H, J = 7.2 Hz, arom.), 7,46 (d, 2H, J = 7.2 Hz, arom.), 9,38 (s, 1H, OH), 10.27 (s, 1H, NH), 11.50 (s, 1H, NH). 13C NMR (100 MHz, DMSO-c/6) 5: 170.7, 167.3, 156.6, 138.4, 137.3, 136.6, 130.3, 129.6, 128.5, 128.3, 128.2, 128.0, 127.1, 126.7, 121.0, 120.5, 56.3, 51.1, 42.1. Anal. Calcd for CzsH^CINzOsSz, % C, 60.66; H, 3.87; N, 5.66. Found, %: C, 60.80; H, 3.80; N, 5.80. rel-(5R, 6S, 7S)-7-(4-Chlorophenyl)-N-(4-hydroxyphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][1:3]thiazole-6-carboxamide (3b) Yield: 57%, mp 220-224°C (EtOH). 1H NMR (400 MHz, DMSO-de) 5: 3.44 (t, 1H, J = 10.4 Hz, 6-H), 4.28 (d, 1H, J = 10.4 Hz, 7-H), 4.68 (d, 1H, J = 10.4 Hz, 5-H), 6.47 (d, 2H, J = 8 .8
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Hz, arom.), 6.60 (d, 2H, J = 8.8 Hz, arom.), 7.00-7.40 (m, 7H, arom.), 7.50 (d, 2H, J = 7.2 Hz, arom.), 9,20 (s, 1H, OH), 9.83 (s, 1H, NH), 11.53 (s, 1H, NH). 13C NMR (100 MHz, DMSO-dg) 5: 170.6, 164.5, 140.2, 139.4, 138.8, 136.3, 129.2, 128.9, 128.6, 128.3, 128.2, 128.1, 127.8, 126.9, 121.6, 114.8, 51.9, 50.8, 46.7. Anal. Calcd for C25 H19CIN2 O3S2 , % C, 60.66; H, 3.87; N, 5.64. Found, %: C, 60.50; H, 3.70; N, 5.70. rel-(5R, 6S, 7S)-7-(4-Methylphenyl)-2-oxo-5-phenyl-N-(4-sulfamoylphenyl)-3,5,6,7tetrahydro-2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3c) Yield: 60%, mp 188-190X (EtOH). 1H NMR (400 MHz, DMSO-d6) ö: 2.27 (s, 3H, CH3), 3.95 (t, 1H, J = 11.4 Hz, 6-H), 4.47 (d, 1H, J = 11.4 Hz, 7-H), 4.73 (d, 1H, J = 11.4 Hz, 5H), 6.98 (d, 2H, J = 7.8 Hz, arom.), 7.09 (d, 2H, J = 7.8 Hz, arom.), 7.28 (s, 2H, NH2), 7.307.50 (m, 5H, arom.), 7.80 (d, 2H, J = 9.0 Hz, arom.), 7.87 (d, 2H, J = 9.0 Hz, arom.), 10.49 (s, 1H, NH), 11.41 (s, 1H, NH). 13C NMR (100 MHz, DMSO-c/6) 5: 167.6, 163.8, 142.0, 140.9, 138.3, 136.6, 134.4, 129.9, 129.0, 127.7, 126.7, 121.7, 120.5, 118.7, 118.4, 104.8, 51.1, 42.3, 42.1, 20.7. Anal. Calcd for C26 H23 N3 O4 S3 , % C, 58.08; H, 4.31; N, 7.82. Found, %: C, 58.20; H, 4.40; N, 7.80. rel-(5R,6S, 7S)-7-(4-Chlorophenyl)-2-oxo-5-phenyl-N-(4-sulfamoylphenyl)-3,5,6,7tetrahydro-2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3d) Yield: 68%, mp 182-184°C (EtOH). 1H NMR (400 MHz, DMSO-dg) 5: 3.95 (t, 1H, J = 11.6 Hz, 6-H), 4.46 (d, 1H, J = 11.6 Hz, 7-H), 4.65 (d, 1H, J = 11.6 Hz, 5-H), 7.08 (d, 2H, J = 8.8 Hz, arom.), 7.27 (d, 2H, J = 8.8 Hz, arom.), 7.29 (s, 2H, NH2), 7.30-7.50 (m, 5H, arom.), 7.76 (d, 2H, J = 8.6 Hz, arom.), 7.83 (d, 2H, J = 8.6 Hz, arom.), 10.56 (s, 1H, NH), 11.55 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 171.0, 167.6, 163.8, 142.0, 140.9, 138.3, 130.5, 129.9, 128.9, 128.4, 127.7, 126.6, 126.5, 121.7, 118.5, 104.1, 56.0, 51.0, 42.1. Anal. Calcd for C25 H20 CIN3O4 S3 , % C, 53.80; H, 3.61; N, 7.53. Found, %: C, 53.70; H, 3.80; N, 7.40. rel-(5R,6S, 7S)-N, 7-Bis(4-chlorophenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3e) Yield: 65%, mp 216-218°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.44 (t, 1H, 10.4 Hz, 6-H), 4.31 (d, 1H, J = 10.4 Hz, 7-H), 4.72 (d, J = 10.4 Hz, 5-H), 6.96 (d, 2H, 8.4 Hz, arom.), 7.03 (d, 2H, J = 8.4 Hz, arom.), 7.16-7.31 (m, 7H, arom.), 7.44 (d, 2H, J - 7.0 Hz, arom.), 9.46 (s, 1H, NH), 11.31 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 170.4, 168.3, 139.2, 136.4, 136.1, 132.1, 131.1, 130.1, 128.5, 128.4, 128.3, 128.2, 127.3, 121.0, 120.4, 107.4, 56.1, 48.4, 44.9. Anal. Calcd for C25 H18CI2 N2O2 S2 , % C, 58.48; H, 3.53; N, 5.46. Found, %: C, 58.30; H, 3.40; N, 5.50. rel-(5R,6S, 7S)-N-(4-Chlorophenyl)-7-(4-methylphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3f) Yield: 75%, mp 200-202°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 2.28 (s, 3H, CH3), 3.44 (t, 1H, J = 10.4 Hz, 6-H), 4.26 (d, 1H, 10.4 Hz, 7-H), 4.70 (d, 1H, J = 10.4 Hz, 5H), 6.96 (d, 2H, J = 8.8 Hz, arom.), 7.02 (d, 2H, J = 8.8 Hz, arom.), 7.05 (d, 2H, J = 7.6 Hz, arom.), 7.13 (d, 2H, J = 7.6 Hz, arom.), 7.18 (t, 1H, J = 7.2 Hz, arom.), 7.24 (t, 2H, J - 7.2 Hz, arom.), 7.44 (d, 2H, J = 7.2 Hz, arom.), 9.41 (s, 1H, NH), 11.23 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 170.5, 168.5, 137.2, 136.7, 136.5, 136.2, 129.0, 128.5, 128.3,
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128.2, 128.1, 127.1, 119.8, 108.3, 56.2, 48.6, 45.2, 20.7. Anal. Calcd for C26H21CIN2O2S2, % C, 63.34; H, 4.29; N, 5.68. Found, %: C, 63.50; H, 4.40; N, 5.70. rel-(5R,6S, 7S)-7-(4-Chlorophenyl)-N-(4-methylphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3g) Yield: 70%, mp 234-236°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 2.16 (s, 3H, CH3), 3.44 (t, 1H, J = 10.4 Hz, 6-H), 4.30 (d, 1H, J = 10.4 Hz, 7-H), 4.70 (d, 1H, J = 10.4 Hz, 5H), 6.75 (d, 2H, J = 8.4 Hz, arom.), 6.83 (d, 2H, J = 8.4 Hz, arom.), 7.20-7.30 (m, 7H, arom.), 7.46 (d, 2H, J = 7.2 Hz, arom.), 9.23 (s, 1H, NH), 11.29 (s, 1H, NH). 13C NMR (100 MHz, DMSO-de) 5: 170.4, 167.9, 139.3, 136.2, 134.9, 132.7, 132.0, 130.2, 128.6, 128.5, 128.4, 128.3, 120.4, 119.8, 119.1, 107.5, 55.8, 48.5, 45.0, 20.3. Anal. Calcd for C26H21CIN2O2S2, % C, 63.34; H, 4.29; N, 5.68. Found, %: C, 63.20; H, 4.40; N, 5.70. rel-(5R,6S, 7S)-N, 7-Bis(4-methylphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3h) Yield: 56%, mp 230-232°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 2.16 (s, 3H, CH3), 2.28 (s, 3H, CH3), 3.45 (t, 1H, J = 10.4 Hz, 6-H), 4.26 (d, 1H, J = 10.4 Hz, 7-H), 4.70 (d, 1H, J = 10.4 Hz, 5-H), 6.77 (d, 2H, J = 8.0 Hz, arom.), 6.82 (d, 2H, J = 8.0 Hz, arom.), 7.06 (d, 2H, J = 7.6 Hz, arom.), 7.14 (d, 2H, J = 7.6 Hz, arom.), 7.21 (t, 1H, J = 7.2 Hz, arom.), 7.25 (t, 2H, J = 7.2 Hz, arom.), 7.46 (d, 2H, J = 7.2 Hz, arom.), 9.18 (s, 1H, NH), 11,22 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 170.5, 168.1, 141.2, 140.1, 137.4, 136.6, 136.4, 135.1, 132.5, 128.9, 128.6, 128.2, 128.1, 119.8, 119.7, 108.5, 55.9, 48.7, 45.2, 20.7, 20.3. Anal. Calcd for C27H24N2O2S2, % C, 68.62; H, 5.12; N, 5.93. Found, %: C, 68.70; H, 5.20; N, 6.00. rel-(5R, 6S, 7S)-N-(4-Chlorophenyl)-2-oxo-5-phenyl-7-(thiophen-2-yl)-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][ 1,3]thiazole-6-carboxamide (3i) Yield: 84%, mp 208-210°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.55 (t, 1H, J = 10.5 Hz, 6-H), 4.72 (d, 1H, J = 10.5 Hz, 7-H), 4.87 (d, J = 10.5 Hz, 5-H), 6.92 (dd, 1H, J = 5.1, 3.6 Hz, thiophen.), 6.98 (d, 1H, J = 2.4 Hz, thiophen.), 7.05 (d, 2H, J = 9.0 Hz, arom.), 7.13 (d, 2H, J = 7.6 Hz, arom.), 7.17 (d, 2H, J = 8.4 Hz, arom.), 7.26 (t, 1H, J = 7.0 Hz, arom.), 7.30 (t, 2H, J = 7.5 Hz, arom.), 7.45 (d, 1H, J = 5.1 Hz, thiophen.), 7.51 (d, 2H, J = 7.2 Hz, arom.), 9.46 (s, 1H, NH), 11.31 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d5) 5: 170.3, 168.3, 142.8, 136.5, 136.0, 128.5, 128.4, 128.3, 128.2, 127.2, 126.7, 126.6, 125.8, 121.0, 119.9, 107.8, 59.6, 56.8, 48.6. Anal. Calcd for C24H24N203S2, % C, 56.95; H, 3.53; N, 5.78. Found, %: C, 56.80; H, 3.70; N, 5.60. rel-(5R, 6S, 7S)-7-(4-Chlorophenyl)-2-oxo-5-phenyi-N-(pyridin-2-yl)-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3j) Yield: 56%, mp 178-180°C (AcOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.48 (t, 1H, d = 10.5 Hz, 6-H), 4.24 (d, 1H, J = 10.5 Hz, 7-H), 4.84 (d, J = 10.5 Hz, 5-H), 7.16-7.45 (m, 9H, arom., pyrid.), 10.21 (s, 1H, NH), 11.50 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 171.7, 169.5, 150.6, 147.5, 139.2, 137.8, 136.1, 132.1, 130.3, 128.7, 128.5, 128.4, 128.3, 120.3, 119.4, 113.0, 107.5, 54.4, 48.6, 45.4. Anal. Calcd for C24Hi8CIN30 2S2, % C, 60.05; H, 3.78; N, 8.75. Found, %: C, 60.10; H, 3.70; N, 8.90.
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rel-(5R, 6S, 7S)-2-Oxo-5-phenyl-N-(pyridin-2-yl)-7-(thiophen-2-yl)-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3k) Yield: 76%, mp 150-152°C (AcOH). 1H NMR (400 MHz, DMSO-ds) 5: 3.43 (t, 1H, J = 10.5 Hz, 6 -H), 4.62 (d, 1H, J = 10.5 Hz, 7-H), 4.84 (d, J = 10.5 Hz, 5-H), 7.20-7.61 (m, 9H, arom., thiophen., pyrid.), 7.86 (d, 1H, J = 4.0 Hz, thiophen.), 8.10-8.20 (m, 2H, pyrid.), 10.29 (s, 1H, NH), 11.47 (s, 1H, NH). 13C NMR (100 MHz, DMSO-cf6) 5: 171.8, 170.4, 150.8, 147.5, 142.5, 137.7, 136.1, 134.6, 129.3, 128.7, 128.5, 128.3, 126.6, 125.9, 119.8, 113.1, 107.9, 56.1,48.8, 47.9. Anal. Calcd for 0 2 2 ^ 7 ^ 0 2 8 3 , % C, 58.51; H, 3.79; N, 9.30. Found, %: C, 58.40; H, 3.90; N, 9.20. rel-(5R, 6S, 7S)-7-(4-Chlorophenyl)-6-(morpholin-4-ylcarbonyl)-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][ 1,3]thiazol-2-one (31) Yield: 90%, mp 206-208°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.44 (t, 1H, J = 10.4 Hz, 6-H), 3.45-3.55 (m, 4H, morpholin), 3.73-3.81 (m, 2H, morpholin), 4.24 (d, 1H, J 10.4 Hz, 7-H), 4.64 (d, 1H, J = 10.4 Hz, 5-H), 7.10 (d, 2H, J = 8.0 Hz, arom.), 7.18-7.34 (m, 7H, arom.), 11.33 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 170.7, 166.8, 138.8, 131.8, 131.0, 128.2, 128.0, 129.9, 127.9, 127.8, 120.9, 104.6, 66.5, 66.1, 56.0, 45.3, 42.9, 41.4. Anal. Calcd for C23 H21 CIN2 O3S2 , % C, 58.40; H, 4.47; N, 5.92. Found, %: C, 58.50; H, 4.30; N, 6.00. rel-(5R, 6S, 7S)-7-(4-Methylphenyl)-6-(morpholin-4-ylcarbonyl)-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][ 1,3]thiazol-2-one (3m) Yield: 77%, mp 176-178°C (PhH). 1H NMR (400 MHz, DMSO-d6) 5: 2.33 (s, 3H, CH3), 2.77-2.86 (m, 4H, morpholin), 2.92-2.95 (m, 2H, morpholin), 3.76 (t , 1H, J = 10.4 Hz, 6 H), 4.14 (d, 1H, J = 10.4 Hz, 7-H), 4.69 (d, 1H, J = 10.4 Hz, 5-H), 7.10 (br.s, 4H, arom.), 7.25-7.34 (m, 3H, arom.), 7.40 (d, 2H, J = 7.0 Hz, arom.), 11.25 (s, 1H, NH). 13C NMR (100 MHz, DMSO-de) 5: 170.5, 168.7, 146.3, 145.3, 136.9, 136.8, 128.9, 128.5, 128.3, 128.2, 120.1, 65.6, 49.2, 48.7, 45.7, 45.3, 41.3, 20.9. Anal. Calcd for C24 H24 N2 O3S2 , % C, 58.40; H, 4.47; N, 5.92. Found, %: C, 58.30; H, 4.50; N, 5.80. Cytotoxic Activity Against Malignant Human Tumor Cells An anticancer in vitro assay was performed on the human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [5-7, 17]. The tested compounds were added to the culture at a single concentration (1CF5 M) and the cultures were incubated for 48 h. Endpoint determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. The growth percentage was evaluated spectrophotometrically versus controls not treated with the test agents.
Acknowledgement We thank Dr. V.L. Narayanan from the Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD, USA, for the in vitro evaluation of anticancer activity. The authors support all people of good will currently struggling for sovereign and unified Ukraine.
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Authors’ Statement Competing Interests The authors declare no conflict of interest.
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Research article
S yn thesis and A n tic a n c e r A c tiv ity of N e w Thiopyrano[2,3-£/]thiazoles Based on C innam ic Acid Am ides Andrii Lozynskyi, Borys Z imenkovsky, Roman Lesyk * Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010, Lviv, Ukraine. * Corresponding author. E-mail: [email protected] (R. Lesyk) Sei Pharm. 2014; 82: 723-733
doi:10.3797/scipharm.1408-05
Published: Accepted:
Received:
September 15ih 2014 September 15th 2014
August 5th 2014
This article is available from: http://dx.doi.org/10.3797/scipharm.1408-05 © Lozynskyi et air, licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.Org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract________________________________________________ Novel /'e/-(5R,6S,7S)-2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2/-/-thiopyrano [2,3-d]thiazole-6-carboxylic acid amides were synthesized in a hetero-D\e\sAlder reaction with a series of cinnamic acid amides. The synthesized compounds were tested for their anticancer activity in vitro in the standard National Cancer Institute 60 cancer cell line assay. Promising compounds 3e, 3g, and 3h with moderate antitumor activity were identified among the synthesized series.
Keywords hefero-Diels-Alder reaction • Cynnamic acid amides Thiopyrano[2,3-cf|[1,3]thiazoles • Anticancer activity
• 5-Ylideneisorhodanines
•
Introduction Investigations of thiopyrano[2,3-c/]thiazole derivatives, the isosteric mimics of biologically active 5-ylidene-4-thiazolidinones, led to the synthesis of compounds with anticancer, antitrypanosomal, and antimycobacterial properties which can provide an opportunity to further study and explore the pharmacological activity of these heterocyclic systems in the future [1-13]. We decided to combine in a single heterocyclic system the thiazolidinone moiety and a fragment of cinnamic acid (Sch. 1). Cinnamic acid and its derivatives exhibit antitumor, antimicrobial, antifungal action and act as histamine H3-receptor antagonists
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[14-16]. Consequently, we have synthesized thiopyrano[2,3-cf]thiazoles using cinnamic acid amides as the dienophile in the reaction of hefero-Diels-Alder. In addition, heterodiene synthesis allows the fixing of the biologically important 4-thiazolidinone fragment in a rigid fused system, preserving its biological activity. Moreover, the combination of thiazole and thiopyran in a fused heterosystem is a precondition for creating ligand-target binding and enhances the potential selectivity to biotargets. This approach suggests the critical impact of the substituent on the biological activity with particular selectivity to various cancer cell lines. Cinnamic acid amides moiety Structure of target compounds
5-Arylidene-4-thiazolidinone moiety
H
Zelisko et al., 2012 [11] Sch. 1 .
0
Anticancer and antimycobacterial activities
Anticancer activity
Atamanyuk et al., 2013 [10]
Lesyk et al., 2006 [4]
Background for the synthesis of target compounds
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Results and Discussion Chemistry The starting 5-aryl(hetaryl)idene-4-thioxo-2-thiazolidinones 1a-d were obtained by the treatm ent of 4-thioxo-2-thiazolidinone with the appropriate aldehydes in glacial acetic acid with a catalytic am ount o f fused sodium acetate [4, 12]. The cinnam ic acid amides were synthesized by the interaction o f the corresponding cinnam ic acid chloride with 4-substituted anilines, morpholine, and 2-am inopyridine in anhydrous dioxane. The heteroDiels-Alder reaction of 2 a -f with 5-aryl(hetaryl)idene-4-thioxo-2-thiazolidinones 1a-d yielded a series of novel re/-(5R,6S,7S)-2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2/-/thiopyrano[2,3-cf]thiazole-6-carboxylic acid amides (Sch. 2).
1a R = 4-OH-C6H4 3a R
1b R = 4-CI-C6H4 1c R = 4-Me-C6H4 1d thiophen-2-yl
4-OH-C6H4, R1= Cl
3b R
4-CI-C6H4, R1 = OH
3c R
4-Me-C6H4, R1 = S 0 2NH2
3d R
4-CI-C6H4, R1 = S 0 2NH2
3e R
4-CI-C6H4, R1 = Cl
3f R
4-Me-C6H4, R1 = Cl
3g R
4-CI-C6H4, R1 = Me
3h R
4-Me-C6H4, R1 = Me
3i R
thiophen-2-yl, R1 = Cl
3j R = 4-CI-C6H4 3I
R = 4-CI-C6H4
3k R = thiophen-2-yl
3m R = 4-Me-C6H4
Sch. 2.
Synthesis of 2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2/-/-thiopyrano[2,3-d]thiazole-6-carboxylic acid amides
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The structure of the synthesized compounds was confirmed by 1H- and 13C NMR. We found the features of the stereochemistry of the above Ziefero-Diels-Alder reaction. Particularly, we have observed that cinnamic acid amides in the [4+2]-cyclocondensation of 5-arylideneisorhodanines form a pair of re/-(5R,6S,7S)-diastereomers. This claim is based on the coupling constant values within 10.4-11.5 Hz and the spectral signals of the thiopyran fragment (triplet and two doublets at 3.40-4.87 ppm), which prove an axial-axial interaction of 5-H, 6-H and 6-H, 7-H proton pairs. Importantly, a similar pattern was observed earlier for cinnamic acids as the dienophile in the reactions of befero-Diels-Alder [
11, 12].
Tab. 1.
Cytotoxic activity of the tested compounds in the concentration 10 60 cancer cell lines
Test Average cpds. growth, % 3b
82.76
3c
101.41
3e
57.09
3g
57.89
3h
77.68
3i
80.64
3j 3k
88.76
3I
100.81
3m
96.07
95.84
Range of growth, %
M against
Most sensitive cell line growth, % (cancer //ne/type) 53.05 (RPMI-8226 / leukemia) 53.05-106.26 61.05 (SF-295 / CNS cancer) 88.61-117.37 88.61 (RXF 393 / renal cancer) 27.11 (MOLT-4 / leukemia) 26.38 (HCT-116 / colon cancer) 32.89 (SF-295 / CNS cancer) 26.38-94.10 35.53 (PC-3 / prostate cancer) 33.44 (MCF7 / breast cancer) 33.81 (T-47D1 breast cancer) 26.51 (MOLT-41 leukemia); 37.02 (RPMI-82261 leukemia) 39.39 (A549/ATCC1 non-small cell lung cancer) 32.09 (HCT-1161 colon cancer) 26.51-91.71 33.18 (SF-2951 CNS cancer) 33.99 (PC-31 prostate cancer) 31.77 (M C F71 breast cancer) 39.88 (T-47D / breast camcer) 30.79 (HOP-921 non-small cell lung cancer) -42.92 (NCI-H5221 non-small cell lung cancer) -42.92-114.10 35.63 (SK-MEL-51 melanoma) -21.73 (CAKI-11 renal cancer) 37.39 (UO-311 renal cancer) 57.75 (SF-295/ CNS cancer) 51.43-119.84 51.43 (PC-3 / prostate cancer) 59.03 (MCF7 / breast cancer) 61.65-112.27 61.65 (SNB-75 / CNS cancer) 72.63-120.48 72.63 (T-47D / breast cancer) 77.27 (SNB-75 1 CNS cancer) 73.58-120.80 78.29 (UO-31 1 renal cancer) 73.58 (T-47D 1 breast cancer) 73.69-110.93 73.69 (SR 1 leukemia);
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Biological Activity The synthesized re/-(5R,6S,7S)-2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2H-thiopyrano[2,3-d]thiazole-6-carboxylic acid amides were selected by the National Cancer Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov) for the in vitro cell line screening to investigate their anticancer activity. Anticancer assays were performed according to the NCI protocol, which is described elsewhere [5-7, 17]. The compounds were evaluated for antitumor activity against 60 cancer lines at a 10 pM concentration. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. The screening results are shown in Table 1. The tested compounds showed different levels of activity on various cancer cell lines. The most active compounds were 3e, 3g, 3h, being highly potent in certain lines of cancer, but they had almost no activity in others. Compounds 3e, 3g have a selective effect on the growth of MOLT-4 (leukemia), HCT-116 (colon cancer), SF-295 (CNS cancer), PC-3 (prostate cancer), MCF7, and T-47D (breast cancer) cancer cell lines in comparison with others.
Sch. 3.
SAR of anticancer potency of the synthesized thiopyrano[2,3-d]thiazole-6carboxylic acids amides
The empirical SAR study (Sch. 3) revealed that: (1) the anticancer activity of the synthesized compounds is sensitive to the nature of the amide fragment in position 6 and substitution in position 7 of the thiopyrano[2,3-d]thiazole moiety;
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(2) introduction of p-Me- or p-CI-C6H4 groups in the amide fragment enhances the potency; (3) the loss of anticancer activity is caused by the introduction of morpholin and pyridine fragments in position 6 or substitution of the arylamide moiety by OH or sulfanilamido groups; (4) synthesized thiopyrano[2,3-c/]thiazole-6-carboxylic acid amides with p-Me- and P-CI-C6H4 groups in postion 7 have the most preferable level of activity compared to other derivatives.
Experimental Chemistry All materials were purchased from Merck, Sigma-Aldrich, or Lancaster and were used without purification. 5-Aryl(hetaryl)idene-4-thioxo-2-thiazolidinones 1a-d were employed as starting materials and prepared according to the method described previously [4, 12]. Melting points were determined in open capillary tubes and were uncorrected. The elemental analyses (C, H, N) were performed using the Perkin-Elmer 2400 CHN analyzer and were within 0.4% of the theoretical values. The 1H- and 13C NMR spectra were recorded on the Varian Gemini 400 MHz or Bruker 125 MHz for frequencies of 100 MHz in DMSO-de using tetramethylsilane as an internal standard. Chemical shifts are reported in ppm units with the use of a 5 scale. The purity of all obtained compounds was checked by 1H-NMR and TLC. General Procedure of the Hetero-Diels-Alder Reaction Affording 3a-m A mixture of appropriate 5-aryl(hetaryl)idene-4-thioxo-2-thiazolidinone (5 mmol) and cinnamic acid amide (5.5 mmol) was refluxed for 4-7 h with a catalytic amount of hydroquinone (2-3 mg) in 15 ml of glacial acetic acid and left overnight at room temperature. The obtained solid products were collected by filtration, washed with water, methanol (5-10 ml), diethyl ether, and recrystallized from the appropriate solvent. rel-(5R, 6S, 7S)-N-(4-Chlorophenyl)-7-(4-hydroxyphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3a) Yield: 59%, mp 234-236°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.47 (t, 1H, J = 10.4 Hz, 6-H), 4.21 (d, 1H, J = 10.4 Hz, 7-H), 4.83 (d, 1H, 10.4 Hz, 5-H), 6.70 (d, 2H, J = 8.8 Hz, arom.), 7.00 (d, 2H, J = 8.8 Hz, arom.), 7.12 (d, 2H, J = 8.8 Hz, arom.), 7.20 (t, 1H, J = 7.2 Hz, arom.), 7.28 (t, 2H, J = 7.2 Hz, arom.), 7,46 (d, 2H, J = 7.2 Hz, arom.), 9,38 (s, 1H, OH), 10.27 (s, 1H, NH), 11.50 (s, 1H, NH). 13C NMR (100 MHz, DMSO-c/6) 5: 170.7, 167.3, 156.6, 138.4, 137.3, 136.6, 130.3, 129.6, 128.5, 128.3, 128.2, 128.0, 127.1, 126.7, 121.0, 120.5, 56.3, 51.1, 42.1. Anal. Calcd for CzsH^CINzOsSz, % C, 60.66; H, 3.87; N, 5.66. Found, %: C, 60.80; H, 3.80; N, 5.80. rel-(5R, 6S, 7S)-7-(4-Chlorophenyl)-N-(4-hydroxyphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][1:3]thiazole-6-carboxamide (3b) Yield: 57%, mp 220-224°C (EtOH). 1H NMR (400 MHz, DMSO-de) 5: 3.44 (t, 1H, J = 10.4 Hz, 6-H), 4.28 (d, 1H, J = 10.4 Hz, 7-H), 4.68 (d, 1H, J = 10.4 Hz, 5-H), 6.47 (d, 2H, J = 8 .8
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Hz, arom.), 6.60 (d, 2H, J = 8.8 Hz, arom.), 7.00-7.40 (m, 7H, arom.), 7.50 (d, 2H, J = 7.2 Hz, arom.), 9,20 (s, 1H, OH), 9.83 (s, 1H, NH), 11.53 (s, 1H, NH). 13C NMR (100 MHz, DMSO-dg) 5: 170.6, 164.5, 140.2, 139.4, 138.8, 136.3, 129.2, 128.9, 128.6, 128.3, 128.2, 128.1, 127.8, 126.9, 121.6, 114.8, 51.9, 50.8, 46.7. Anal. Calcd for C25 H19CIN2 O3S2 , % C, 60.66; H, 3.87; N, 5.64. Found, %: C, 60.50; H, 3.70; N, 5.70. rel-(5R, 6S, 7S)-7-(4-Methylphenyl)-2-oxo-5-phenyl-N-(4-sulfamoylphenyl)-3,5,6,7tetrahydro-2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3c) Yield: 60%, mp 188-190X (EtOH). 1H NMR (400 MHz, DMSO-d6) ö: 2.27 (s, 3H, CH3), 3.95 (t, 1H, J = 11.4 Hz, 6-H), 4.47 (d, 1H, J = 11.4 Hz, 7-H), 4.73 (d, 1H, J = 11.4 Hz, 5H), 6.98 (d, 2H, J = 7.8 Hz, arom.), 7.09 (d, 2H, J = 7.8 Hz, arom.), 7.28 (s, 2H, NH2), 7.307.50 (m, 5H, arom.), 7.80 (d, 2H, J = 9.0 Hz, arom.), 7.87 (d, 2H, J = 9.0 Hz, arom.), 10.49 (s, 1H, NH), 11.41 (s, 1H, NH). 13C NMR (100 MHz, DMSO-c/6) 5: 167.6, 163.8, 142.0, 140.9, 138.3, 136.6, 134.4, 129.9, 129.0, 127.7, 126.7, 121.7, 120.5, 118.7, 118.4, 104.8, 51.1, 42.3, 42.1, 20.7. Anal. Calcd for C26 H23 N3 O4 S3 , % C, 58.08; H, 4.31; N, 7.82. Found, %: C, 58.20; H, 4.40; N, 7.80. rel-(5R,6S, 7S)-7-(4-Chlorophenyl)-2-oxo-5-phenyl-N-(4-sulfamoylphenyl)-3,5,6,7tetrahydro-2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3d) Yield: 68%, mp 182-184°C (EtOH). 1H NMR (400 MHz, DMSO-dg) 5: 3.95 (t, 1H, J = 11.6 Hz, 6-H), 4.46 (d, 1H, J = 11.6 Hz, 7-H), 4.65 (d, 1H, J = 11.6 Hz, 5-H), 7.08 (d, 2H, J = 8.8 Hz, arom.), 7.27 (d, 2H, J = 8.8 Hz, arom.), 7.29 (s, 2H, NH2), 7.30-7.50 (m, 5H, arom.), 7.76 (d, 2H, J = 8.6 Hz, arom.), 7.83 (d, 2H, J = 8.6 Hz, arom.), 10.56 (s, 1H, NH), 11.55 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 171.0, 167.6, 163.8, 142.0, 140.9, 138.3, 130.5, 129.9, 128.9, 128.4, 127.7, 126.6, 126.5, 121.7, 118.5, 104.1, 56.0, 51.0, 42.1. Anal. Calcd for C25 H20 CIN3O4 S3 , % C, 53.80; H, 3.61; N, 7.53. Found, %: C, 53.70; H, 3.80; N, 7.40. rel-(5R,6S, 7S)-N, 7-Bis(4-chlorophenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3e) Yield: 65%, mp 216-218°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.44 (t, 1H, 10.4 Hz, 6-H), 4.31 (d, 1H, J = 10.4 Hz, 7-H), 4.72 (d, J = 10.4 Hz, 5-H), 6.96 (d, 2H, 8.4 Hz, arom.), 7.03 (d, 2H, J = 8.4 Hz, arom.), 7.16-7.31 (m, 7H, arom.), 7.44 (d, 2H, J - 7.0 Hz, arom.), 9.46 (s, 1H, NH), 11.31 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 170.4, 168.3, 139.2, 136.4, 136.1, 132.1, 131.1, 130.1, 128.5, 128.4, 128.3, 128.2, 127.3, 121.0, 120.4, 107.4, 56.1, 48.4, 44.9. Anal. Calcd for C25 H18CI2 N2O2 S2 , % C, 58.48; H, 3.53; N, 5.46. Found, %: C, 58.30; H, 3.40; N, 5.50. rel-(5R,6S, 7S)-N-(4-Chlorophenyl)-7-(4-methylphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3f) Yield: 75%, mp 200-202°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 2.28 (s, 3H, CH3), 3.44 (t, 1H, J = 10.4 Hz, 6-H), 4.26 (d, 1H, 10.4 Hz, 7-H), 4.70 (d, 1H, J = 10.4 Hz, 5H), 6.96 (d, 2H, J = 8.8 Hz, arom.), 7.02 (d, 2H, J = 8.8 Hz, arom.), 7.05 (d, 2H, J = 7.6 Hz, arom.), 7.13 (d, 2H, J = 7.6 Hz, arom.), 7.18 (t, 1H, J = 7.2 Hz, arom.), 7.24 (t, 2H, J - 7.2 Hz, arom.), 7.44 (d, 2H, J = 7.2 Hz, arom.), 9.41 (s, 1H, NH), 11.23 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 170.5, 168.5, 137.2, 136.7, 136.5, 136.2, 129.0, 128.5, 128.3,
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128.2, 128.1, 127.1, 119.8, 108.3, 56.2, 48.6, 45.2, 20.7. Anal. Calcd for C26H21CIN2O2S2, % C, 63.34; H, 4.29; N, 5.68. Found, %: C, 63.50; H, 4.40; N, 5.70. rel-(5R,6S, 7S)-7-(4-Chlorophenyl)-N-(4-methylphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3g) Yield: 70%, mp 234-236°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 2.16 (s, 3H, CH3), 3.44 (t, 1H, J = 10.4 Hz, 6-H), 4.30 (d, 1H, J = 10.4 Hz, 7-H), 4.70 (d, 1H, J = 10.4 Hz, 5H), 6.75 (d, 2H, J = 8.4 Hz, arom.), 6.83 (d, 2H, J = 8.4 Hz, arom.), 7.20-7.30 (m, 7H, arom.), 7.46 (d, 2H, J = 7.2 Hz, arom.), 9.23 (s, 1H, NH), 11.29 (s, 1H, NH). 13C NMR (100 MHz, DMSO-de) 5: 170.4, 167.9, 139.3, 136.2, 134.9, 132.7, 132.0, 130.2, 128.6, 128.5, 128.4, 128.3, 120.4, 119.8, 119.1, 107.5, 55.8, 48.5, 45.0, 20.3. Anal. Calcd for C26H21CIN2O2S2, % C, 63.34; H, 4.29; N, 5.68. Found, %: C, 63.20; H, 4.40; N, 5.70. rel-(5R,6S, 7S)-N, 7-Bis(4-methylphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3h) Yield: 56%, mp 230-232°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 2.16 (s, 3H, CH3), 2.28 (s, 3H, CH3), 3.45 (t, 1H, J = 10.4 Hz, 6-H), 4.26 (d, 1H, J = 10.4 Hz, 7-H), 4.70 (d, 1H, J = 10.4 Hz, 5-H), 6.77 (d, 2H, J = 8.0 Hz, arom.), 6.82 (d, 2H, J = 8.0 Hz, arom.), 7.06 (d, 2H, J = 7.6 Hz, arom.), 7.14 (d, 2H, J = 7.6 Hz, arom.), 7.21 (t, 1H, J = 7.2 Hz, arom.), 7.25 (t, 2H, J = 7.2 Hz, arom.), 7.46 (d, 2H, J = 7.2 Hz, arom.), 9.18 (s, 1H, NH), 11,22 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 170.5, 168.1, 141.2, 140.1, 137.4, 136.6, 136.4, 135.1, 132.5, 128.9, 128.6, 128.2, 128.1, 119.8, 119.7, 108.5, 55.9, 48.7, 45.2, 20.7, 20.3. Anal. Calcd for C27H24N2O2S2, % C, 68.62; H, 5.12; N, 5.93. Found, %: C, 68.70; H, 5.20; N, 6.00. rel-(5R, 6S, 7S)-N-(4-Chlorophenyl)-2-oxo-5-phenyl-7-(thiophen-2-yl)-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][ 1,3]thiazole-6-carboxamide (3i) Yield: 84%, mp 208-210°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.55 (t, 1H, J = 10.5 Hz, 6-H), 4.72 (d, 1H, J = 10.5 Hz, 7-H), 4.87 (d, J = 10.5 Hz, 5-H), 6.92 (dd, 1H, J = 5.1, 3.6 Hz, thiophen.), 6.98 (d, 1H, J = 2.4 Hz, thiophen.), 7.05 (d, 2H, J = 9.0 Hz, arom.), 7.13 (d, 2H, J = 7.6 Hz, arom.), 7.17 (d, 2H, J = 8.4 Hz, arom.), 7.26 (t, 1H, J = 7.0 Hz, arom.), 7.30 (t, 2H, J = 7.5 Hz, arom.), 7.45 (d, 1H, J = 5.1 Hz, thiophen.), 7.51 (d, 2H, J = 7.2 Hz, arom.), 9.46 (s, 1H, NH), 11.31 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d5) 5: 170.3, 168.3, 142.8, 136.5, 136.0, 128.5, 128.4, 128.3, 128.2, 127.2, 126.7, 126.6, 125.8, 121.0, 119.9, 107.8, 59.6, 56.8, 48.6. Anal. Calcd for C24H24N203S2, % C, 56.95; H, 3.53; N, 5.78. Found, %: C, 56.80; H, 3.70; N, 5.60. rel-(5R, 6S, 7S)-7-(4-Chlorophenyl)-2-oxo-5-phenyi-N-(pyridin-2-yl)-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3j) Yield: 56%, mp 178-180°C (AcOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.48 (t, 1H, d = 10.5 Hz, 6-H), 4.24 (d, 1H, J = 10.5 Hz, 7-H), 4.84 (d, J = 10.5 Hz, 5-H), 7.16-7.45 (m, 9H, arom., pyrid.), 10.21 (s, 1H, NH), 11.50 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 171.7, 169.5, 150.6, 147.5, 139.2, 137.8, 136.1, 132.1, 130.3, 128.7, 128.5, 128.4, 128.3, 120.3, 119.4, 113.0, 107.5, 54.4, 48.6, 45.4. Anal. Calcd for C24Hi8CIN30 2S2, % C, 60.05; H, 3.78; N, 8.75. Found, %: C, 60.10; H, 3.70; N, 8.90.
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rel-(5R, 6S, 7S)-2-Oxo-5-phenyl-N-(pyridin-2-yl)-7-(thiophen-2-yl)-3,5,6,7-tetrahydro-2Hthiopyrano[2,3-d][1,3]thiazole-6-carboxamide (3k) Yield: 76%, mp 150-152°C (AcOH). 1H NMR (400 MHz, DMSO-ds) 5: 3.43 (t, 1H, J = 10.5 Hz, 6 -H), 4.62 (d, 1H, J = 10.5 Hz, 7-H), 4.84 (d, J = 10.5 Hz, 5-H), 7.20-7.61 (m, 9H, arom., thiophen., pyrid.), 7.86 (d, 1H, J = 4.0 Hz, thiophen.), 8.10-8.20 (m, 2H, pyrid.), 10.29 (s, 1H, NH), 11.47 (s, 1H, NH). 13C NMR (100 MHz, DMSO-cf6) 5: 171.8, 170.4, 150.8, 147.5, 142.5, 137.7, 136.1, 134.6, 129.3, 128.7, 128.5, 128.3, 126.6, 125.9, 119.8, 113.1, 107.9, 56.1,48.8, 47.9. Anal. Calcd for 0 2 2 ^ 7 ^ 0 2 8 3 , % C, 58.51; H, 3.79; N, 9.30. Found, %: C, 58.40; H, 3.90; N, 9.20. rel-(5R, 6S, 7S)-7-(4-Chlorophenyl)-6-(morpholin-4-ylcarbonyl)-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][ 1,3]thiazol-2-one (31) Yield: 90%, mp 206-208°C (EtOH). 1H NMR (400 MHz, DMSO-d6) 5: 3.44 (t, 1H, J = 10.4 Hz, 6-H), 3.45-3.55 (m, 4H, morpholin), 3.73-3.81 (m, 2H, morpholin), 4.24 (d, 1H, J 10.4 Hz, 7-H), 4.64 (d, 1H, J = 10.4 Hz, 5-H), 7.10 (d, 2H, J = 8.0 Hz, arom.), 7.18-7.34 (m, 7H, arom.), 11.33 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) 5: 170.7, 166.8, 138.8, 131.8, 131.0, 128.2, 128.0, 129.9, 127.9, 127.8, 120.9, 104.6, 66.5, 66.1, 56.0, 45.3, 42.9, 41.4. Anal. Calcd for C23 H21 CIN2 O3S2 , % C, 58.40; H, 4.47; N, 5.92. Found, %: C, 58.50; H, 4.30; N, 6.00. rel-(5R, 6S, 7S)-7-(4-Methylphenyl)-6-(morpholin-4-ylcarbonyl)-5-phenyl-3,5,6,7-tetrahydro2H-thiopyrano[2,3-d][ 1,3]thiazol-2-one (3m) Yield: 77%, mp 176-178°C (PhH). 1H NMR (400 MHz, DMSO-d6) 5: 2.33 (s, 3H, CH3), 2.77-2.86 (m, 4H, morpholin), 2.92-2.95 (m, 2H, morpholin), 3.76 (t , 1H, J = 10.4 Hz, 6 H), 4.14 (d, 1H, J = 10.4 Hz, 7-H), 4.69 (d, 1H, J = 10.4 Hz, 5-H), 7.10 (br.s, 4H, arom.), 7.25-7.34 (m, 3H, arom.), 7.40 (d, 2H, J = 7.0 Hz, arom.), 11.25 (s, 1H, NH). 13C NMR (100 MHz, DMSO-de) 5: 170.5, 168.7, 146.3, 145.3, 136.9, 136.8, 128.9, 128.5, 128.3, 128.2, 120.1, 65.6, 49.2, 48.7, 45.7, 45.3, 41.3, 20.9. Anal. Calcd for C24 H24 N2 O3S2 , % C, 58.40; H, 4.47; N, 5.92. Found, %: C, 58.30; H, 4.50; N, 5.80. Cytotoxic Activity Against Malignant Human Tumor Cells An anticancer in vitro assay was performed on the human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [5-7, 17]. The tested compounds were added to the culture at a single concentration (1CF5 M) and the cultures were incubated for 48 h. Endpoint determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. The growth percentage was evaluated spectrophotometrically versus controls not treated with the test agents.
Acknowledgement We thank Dr. V.L. Narayanan from the Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD, USA, for the in vitro evaluation of anticancer activity. The authors support all people of good will currently struggling for sovereign and unified Ukraine.
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Authors’ Statement Competing Interests The authors declare no conflict of interest.
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